This application claims the benefit of priority under 35 USC 119(a)-(d) to Republic of Korea application 10-2001-86765, filed Dec. 28, 2001.
1. Field of the Invention
The present invention relates to the field of biotechnology and especially antisense therapy using closed covalent antisense molecule that is targeted to TGF-β. The invention also relates to a method of delivering the antisense molecule to a cell. The invention further relates to a method of treating diseases caused by the production of TGF-β.
2. General Background and State of the Art
Renal tubulointerstitial fibrosis, characterized by the accumulation of extracellular matrix proteins (ECMs), is a common consequence of progressive renal diseases (Kuncio et al., Kidney Int. 39, 550-556(1991)). Obstructed kidney with unilateral ureteric obstruction (UUO) is a well established animal model system for renal injury accompanied by tubulointerstitial fibrosis (Klahr et al., Kidney Int. 54, 286-300(1998)). The mechanical disturbance resulting from ureteral ligation leads to hydronephrosis, loss of renal parenchyma, and tubular changes such as dilation, atrophy, and apoptosis (Gonzalez-Avila et al., Pathobiology 66, 196-204(1998); Truong et al., Kidney Int. 50, 200-207(1996)). Although the mechanisms underlying the progression of tubulointerstitial fibrosis have not been fully elucidated, numerous cytokines have been implicated as mediators of tubulointerstitial fibrosis in kidneys with UUO. Among these cytokines, transforming growth factor-β1 (TGF-β1) plays an important role in renal fibrosis, as evidenced by glomerulosclerosis and tubulointerstitial fibrosis (Schiffer et al., J. Clin. Invest. 108, 807-816(2001); Wang et al., Kidney Int. 60, 96-105(2001)). TGF-β1 is involved in accumulation of ECMs responding to tissue injury for normal repair and is responsible for fibrotic changes by aberrant overproduction of ECMs such as proteoglycans, collagens, fibronectin, and glycoproteins (Branton et al., Microbes Infect. 1, 1349-1365(1999); Okuda et al., J. Clin. Invest. 86, 453-462(1990)). TGF-β1 also inhibits the degradation of newly synthesized matrix protein by upregulating the synthesis of protease inhibitors and downregulating the synthesis of matrix-degrading proteases. Thus, effective blockade of TGF-β1 synthesis or action appears to be a promising method for preventing fibrotic conditions as suggested by several reports (Border et al., Nature 360, 361-364(1992); Akagi et al., Kidney Int. 50, 148-155(1996); Isaka et al., Nat. Med. 2, 418-423(1996); Isaka et al., Kidney Int. 55, 465-475(1999)).
Antisense oligonucleotides (AS oligos) have been valuable in the functional study of gene products by reducing expression of genes in a sequence-specific manner. However, the use of oligos still has several critical problems such as instability to nuclease, sequence nonspecificity and poor cellular uptake. Various chemically modified oligos such as phosphorothioate and methylphosphonate oligos have been developed to augment stability against nucleases. However, each of the modified oligonucleotides exhibits its own problems, which include lack of sequence specificity, insensitivity to RNaseH and prolongation of partial thrombosis time. Further, there has been fear that recycled hydrolyzed modified nucleotides may be incorporated into the genome during DNA repair and replication, causing mutations in genomic DNA. We previously reported that ribbon-type antisense (RiAS) oligos with a covalently closed structure were very stable and effective in specific ablation of target c-myb mRNA, and had few of the problems associated with other modified AS oligos (Moon et al., J. Biol. Chem. 275, 4647-4653(2000); and PCT/KR00/00305), which are incorporated by reference in their entirety.
U.S. Pat. No. 5,683,874 discloses forming a covalently closed type nucleic acid sequence but requires that a parallel and anti-parallel nucleic acid sequence be present on the opposite sides of the loop to form a triple helical structure. However, such structure is useful mainly for binding genomic promoter region and not for binding complementary mRNA for targeted degradation by RNase H. Thus, the '874 patent fails to disclose or suggest that a covalently closed antisense structure with or without such parallel and anti-parallel sequences may be effective in ablating target nucleic acid expression.
Cellular uptake of antisense oligos can be enhanced by forming complexes with liposomes. Although liposomes have several advantages such as low toxicity, lack of immunogenicity, and simplicity of production, liposomes exhibit relatively poor cellular uptake. It has been shown that a protein fused with the protein transduction domain of the tat polypeptide of human immunodeficiency virus (HIV) can be efficiently delivered to all tissues in mice, including the brain (Schwarze et al., Science 285, 1569-1572(1999)). The tat peptide covalently harnessed on the surface of liposomes increases intracellular delivery (Torchilin et al., Proc. Natl. Acad. Sci. USA 98, 8786-8791(2001)). Further, a small region of the tat protein, residues from 49 to 57 containing 2 lysines and 6 arginines, has nuclear localization property.
There is a continuing need to make therapeutic antisense molecules that are specific, safe and efficacious, as well as a system for delivering the antisense molecules effectively.